Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O

Abstract

Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 M urea, 1% Triton X-100, 30% sucrose, 0.02% NaN3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 M guanidinium chloride and 3 M CsCl reduced the sensitivity of the assay to 30-50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.